Generation of High-Value Genomic Resource in Rice: A "Subgenomic Library" of Low-Light Tolerant Rice Cultivar Swarnaprabha

Abstract

Rice is the major staple food crop for more than 50% of the world's total population, and its production is of immense importance for global food security. As a photophilic plant, its yield is governed by the quality and duration of light. Like all photosynthesizing plants, rice perceives the changes in the intensity of environmental light using phytochromes as photoreceptors, and it initiates a morphological response that is termed as the shade-avoidance response (SAR). Phytochromes (PHYs) are the most important photoreceptor family, and they are primarily responsible for the absorption of the red (R) and far-red (FR) spectra of light. In our endeavor, we identified the morphological differences between two contrasting cultivars of rice: IR-64 (low-light susceptible) and Swarnaprabha (low-light tolerant), and we observed the phenological differences in their growth in response to the reduced light conditions. In order to create genomic resources for low-light tolerant rice, we constructed a subgenomic library of Swarnaprabha that expedited our efforts to isolate light-responsive photoreceptors. The titer of the library was found to be 3.22 × 10⁵ cfu/mL, and the constructed library comprised clones of 4-9 kb in length. The library was found to be highly efficient as per the number of recombinant clones. The subgenomic library will serve as a genomic resource for the Gramineae community to isolate photoreceptors and other genes from rice.

Keywords: Gramineae; far-red light; light-responsive genes; photoreceptors; phytochromes; rice.

Figure 1

Mode of action of phytochromes in light perception in plants. The inactive Pr form in cytosol is converted to an active Pfr form by the absorption of red light, which, upon irradiation, moves to the nucleus to interact with numerous cell components to cause physiological changes in response to light. The Pfr form of phytochromes can absorb far-red light and then return to the Pr form, which implies a Pr/Pfr photo-reversibility, in which phytochromes operate as toggle switches that turn on with R light and off with FR light.

Figure 2

Structure of plant phytochrome. The photosensory module (PSM) of the phytochrome is predominantly made up of the conserved domains PAS, GAF, and PHY, which are responsible for photosensory activity and bilin molecule binding. The PAS and GAF domains of the photosensory core (the N-terminus) are knotted together. The PHY domain is responsible for stabilizing the Pfr chromophore. The distant end of the protein (i.e., the C-terminus), also called the output module (OPM), is made up of two domains: the PAS-A and PAS-B sub-domains (together called the PRD–PAS repeat domain), as well as the histidine kinase-related domain (HKRD), and it is responsible for dimerization. The PAS-A and PAS-B sub-domains are only found in plant phytochromes, and they form the PAS-repeat domain (PRD), whereas the HKRD domain is also found in phytochrome-like proteins.

Figure 3

Morphologies ofIR-64 and Swarnaprabha 21-day-old seedlings grown in light-controlled growth chambers.

Figure 4

Growth parameters of contrasting rice cultivars. (a) 10 individual plants each of Swarnaprabha and IR-64, in response to low-light. (b) The average height of the plants of Swarnaprabha (20.76 cm) was found to be higher in comparison with that of IR-64 (16.18 cm).

Figure 5

Isolation of genomic DNA from contrasting rice cultivars (IR-64 and Swarnaprabha). Lane M: Lambda DNA/EcoRI + HindIII ladder. The concentration of isolated genomic DNA in both samples, IR-64 (5 lanes) and Swarnaprabha (5 lanes), was found to be in a range of 500–800 ng/μL.

Figure 6

Partial restriction profile of Swarnaprabha genomic DNA. (A) Standardization of restriction of genomic DNA with EcoRI was carried out for 20′ (Lane 1), 40′ (Lane 2), 60′ (Lane 3), 90′ (Lane 4), and 120′ (Lane 5) durations. (B) Excised genomic size of 4–9 kb for ligation into pBluescript vector.

Figure 7

Blue–white screening of recombinant clones representing subgenomic library of Swarnaprabha by α-complementation. White colonies are recombinant clones containing subgenomic DNA of rice. Blue colonies are non-recombinant vector molecules (self-ligated pBluescript DNA).

Figure 8

Axenic culture of subgenomic library of rice. Putative recombinant clones (white colonies) were restreaked on LA+ carbenicillin (100 μg/mL) grid plates supplemented with X-Gal +IPTG for α-complementation. Bonafide white colonies (representing recombinant clones) after two rounds of sub-culturing were stored individually for long-term storage.

Figure 9

Colony PCR of randomly picked white colonies with universal M13 forward and reverse primers. Out of several colonies screened, sixty-six positive colonies were found to harbor different insert sizes. Lane M is the marker DNA of the indicated size, and Lanes 69–157 are the sixty-six positive clones.

Figure 10

Colony PCR of randomly picked white colonies with gene-specific phyA forward and reverse primers. Out of 200 colonies screened, 35 positive colonies were found to harbor phyA genes (similar sizes but varied intensities). Lane M is the marker DNA of the indicated size, and Lanes 1–35 are thirty-five positive clones.